Team DaNA Presents:

Novel one-step method for DNA origami continuous production using nicking restriction enzyme and strand displacement amplification

Introduction

DNA origami has been a versatile technique in nucleic acid nanotechnology. However, the current DNA origami procedure is inconvenient for the construction of complex structures, which requires extensive pipetting. This would be problematic for the production of complex DNA nanostructures and limit the versatility of DNA origami.

Motivated by the necessity of a simpler and faster procedure, our proposed method alleviates the need of tedious pipetting in making a DNA nanostructure. Instead of synthesizing hundreds or thousands of staple strands, we used a single long DNA strand that combines of the required staple sequences, which then be cut into the staples. Our procedure enables a faster and more precise construction of complex DNA nanostructures.

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Since its introduction in 2006, DNA origami has brought major advancement in DNA nanotechnology. The technique allows easy designing and construction of 2D and 3D nanostructures by making use of DNA’s self-assembly property. Its simple principle of “folding” a single DNA strand (the scaffold) with the help of shorter oligonucleotides (the staples) quickly attracted the attention of nanotechnologists. Although it’s initially performed for aesthetic purposes or proof-of-concept researches, applications of DNA origami structures rapidly emerged. Now, DNA origami has been widely utilized to create various functional nanostructures with ever-increasing complexity.

However, production of complex structures using the current DNA origami procedure face significant challenges. Larger and more complex DNA origami structures require a large amount of staple strands. With the current method, each sequence of staple strands need to be synthesized separately before being mixed with the scaffold strands. For structures with hundreds of staple sequences, the synthesis process would be troublesome since many different kinds of oligonucleotides are required. Furthermore, the current method also requires thermal annealing reactions, which takes a lot of time to produce the desired structures. Therefore, the application of DNA origami technique to build complex structures is currently limited. In order to efficiently build complex DNA nanostructures, a new method to perform DNA origami is necessary.

Idea

We propose a novel idea of preparing DNA origami staple strands that avoids the need of creating multiple different staples and does not require a thermal cycler. Instead of synthesizing each staple sequence separately, we combined all of them into a single monolithic DNA strand. The strand consists of the required staple sequences spaced by restriction sites. Combining all of the staples into one long strand alleviates the need of extensive pipetting.

In our project, we utilized three types of enzyme to aid with the preparation of staples: restriction enzyme, nicking endonuclease, and polymerase. Restriction enzyme cuts our monolithic strand into the component staple strands in the mixture. Afterwards, the resulting staple strands are amplified with the help of nicking endonuclease and polymerase through strand displacement amplification, which is an isothermal process (i.e. does not require thermal cycling). Combining all of the enzymes in the scaffold and monolithic strand mixture would produce the staple strands that will readily anneal with the scaffolds. In other words, aside from reducing the need of pipetting, our method also provides a one-step reaction of producing a DNA origami structure.

Relevance

The relevance of our method lies in its ability to build complex DNA origami nanostructures with relative ease. Compared to the conventional DNA origami procedure, our method allows a convenient and quick construction of large and intricate nanostructures. In this regard, our method could revolutionize DNA nanotechnology by enabling the creation of DNA nanostructures that previously hard to build.

Feasibility

The procedure we outlined provides a feasible means of creating complicated DNA nanostructures. Compared to the conventional method, our procedure could be carried out faster since it combines the staple amplification and annealing procedures into a single reaction. To produce a complex structure with hundreds of staples, our proposed method is more feasible compared to the current DNA origami method.

Merit

Our proposed method offers a simple and elegant solution for building large and complicated DNA nanostructures. In our procedure, the separate synthesis of multiple DNA staples is no longer needed; it is replaced by cutting a long DNA strand into the required staples. The utilization of strand displacement amplification also renders thermal cycling unneeded. Thus, we simplified the procedure of DNA origami into an elegant one-step reaction.

method

1. Preparation of the monolithic DNA strand
The monolithic strand consisting of the necessary staple sequences and restriction sites (“biter sequences”) was assembled via Golden Gate assembly method; we used BspMI restriction sites as the biter sequence. The assembly was performed using BsmBI restriction enzyme and T7 ligase in T4 ligase buffer solution. Propagation of the resulting strand was carried out by inserting it into pSB1C3 cloning vector, which then transformed into TOP10 Escherichia coli. The plasmids were then isolated from the host to be used for the DNA origami procedure.
2. One-step reaction for performing DNA origami
To perform a one-step reaction for building DNA nanostructures, we utilized the pSB1C3 vector as scaffold strand. The plasmid was first mixed with BspMI restriction enzyme to cut the inserted monolithic strand in the plasmid into staple strands. We then utilized Bst 2.0 - a strand-displacing DNA polymerase - and Nt.BspQ1 nicking enzyme to multiply the staples via strand displacement amplification. The nicking enzyme introduced a nick in the cut double-stranded strands, which induced Bst 2.0 to initiate replication from the nicked site. In the process, the polymerase displaced the nicked strands, thus producing single-stranded staples. Meanwhile, the scaffold strands were amplified via primer elongation, also by Bst 2.0. Ultimately, the generated staples bonded at their corresponding sites in the scaffold with the similar mechanism to conventional DNA origami technique, producing our desired structure.
3. Visualization and characterization of the nanostructures via TEM

results

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Team Photo

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Tetrahedron

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DNA Cup Deformed Shape View 1

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DNA Cup Deformed Shape View 2

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DNA Cup Deformed Shape View 3

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DNA Cup Helix Perspective

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DNA Cup Side 1

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DNA Cup Side 2

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DNA Cup Ring Side

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DNA Cup Ring Top

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DNA Cup RMSF 1

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DNA Cup RMSF 2

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DNA Cup RMSF 3

Future

We envision a future where DNA origami is the leading force in the field of nanotechnology. To achieve that goal, a simple and effective procedure of building DNA nanostructures with DNA origami is necessary. We believe our proposed method could go beyond the limitations of current techniques and pave a way to the creation of intricate nanostructures with beneficial functions.